Malignant melanoma is one of the most aggressive types of human cancers. Its ability to metastasize in combination with its notorious resistance to conventional chemotherapeutical agents makes melanoma extremely difficult to cure. Consequently, the median survival of patients with metastatic melanoma is only 8.5 months. The acquisition of invasion capability, which occurs already in primary melanomas, is a prerequisite for metastasis and is considered a critical event associated with poor prognosis. We have recently demonstrated that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in the de novo biosynthesis of purine nucleotides, was downregulated at the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade (in vitro and in vivo), and grow as tumor xenografts. We further demonstrated that depletion of guanosine monophosphate synthase (GMPS), a functional antagonist of GMPR, decreases active (GTP-bound) RAC1, RHOA and RHOC. We hypothesized that GMPS, GMPR, and perhaps other guanylate biosynthesis enzymes, regulate the activity of the above RHO-GTPases via modulation of GTP levels in the vicinity of these RHO-GTPases. This hypothesis will be tested in Specific Aim 1. We demonstrated that the activity of GMPR can be regulated by phosphorylation. Unbiased in vitro kinase screening identified several kinases as potential candidates for GMPR phosphorylation. Modulation of GTP levels has never been considered as a mechanism of regulation of invasion by any kinase. Therefore, in Specific Aim 2, using functional approaches, we will test candidates' ability to regulate GTP levels, RHO- GTPase activity and cell invasion in a GMPR-dependent manner. Currently, no efficient chemotherapy exists for melanoma patients with wildtype BRAF and mutant NRAS. Survival of patients with mutant BRAF was improved by the introduction of its inhibitor vemurafenib (VEM), however rapidly developing resistance circumvents VEM efficacy. Although the mechanisms of such resistance vary, several melanoma cell lines independently selected for VEM resistance possessed increased invasion ability. We demonstrated that GMPS plays a major role in the invasion and tumorigenicity of cells derived from either BRAFV600E or NRASQ61R human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared to primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice in vivo. In Specific Aim 3, we will evaluate the efficacy of AGM alone or in combination with VEM in several preclinical melanoma models.